Real-time and high throughput monitoring of cAMP in live cells using a fluorescent membrane potential-sensitive dye.
Real-time and high throughput monitoring of cAMP in live cells using a fluorescent membrane potential-sensitive dye.
复制标题
使用荧光膜电位敏感染料实时高通量监测活细胞中的 cAMP。
DOI：
10.1089/adt.2006.4.461
复制
发表时间：
2006
影响因子：
1.8
通讯作者：
Diwu,Zhenjun
中科院分区：
文献类型：
--
作者：
Tang,Yi;Li,Xiuqing;He,Jianjun;Lu,Junge;Diwu,Zhenjun
腺苷-3',5'-环单磷酸腺苷（cAMP）传递来自g蛋白偶联受体（gpcr）的信号并调节多种下游细胞事件。然而，很少有可靠的分析方法可用于测量活细胞中的cAMP。大多数现有的cAMP测定需要细胞裂解和/或具有相对较低的通量。我们报告了一种基于活细胞的cAMP测定方法，该方法已被开发用于记录细胞内cAMP的实时变化。通过采用突变的环核苷酸门控离子通道（CNGC）作为cAMP生物传感器，cAMP水平的变化与跨膜电位的变化耦合，通过一种新的荧光膜电位（MP）敏感染料HLB 021-152测量。我们已经成功地使用HLB 021-152在含血清和无血清环境下均匀监测活细胞中的cAMP刺激。用激动剂刺激cngc克隆的人胚胎肾293细胞内源性或异质gpcr后，HLB 021-152的荧光信号迅速增加。它比现有的测定细胞MP的“金标准”染料DiSBAC2(3)具有更大的测定动态范围。这种新的MP染料可以很容易地配制成高通量筛选GPCR调节剂，无论有血清或无血清。
Adenosine-3',5'-cyclic monophosphate (cAMP) conveys the signals from G-protein coupled receptors (GPCRs) and regulates a variety of downstream cellular events. However, there are few robust assays available for measuring cAMP in live cells. Most of the existing cAMP assays require cell lysis and/or have relatively low throughput. We report a live cell-based cAMP assay that has been developed to record the real-time changes in intracellular cAMP. By employing a mutated cyclic-nucleotide-gated ion channel (CNGC) as the cAMP biosensor, the change of cAMP level is coupled to the change of transmembrane potential that is measured through a new fluorescent membrane potential (MP)-sensitive dye, HLB 021-152. We have successfully used HLB 021-152 for homogeneously monitoring cAMP stimulations in live cells under both serum-containing and serumfree environments. Upon stimulating the endogenous or heterogenous GPCRs on CNGC-cloned human embryonic kidney 293 cells with agonists, the fluorescence signal of HLB 021-152 increases rapidly. It has much greater assay dynamic range than DiSBAC2(3), the existing "gold standard" dye for measuring cellular MP. This new MP dye can be readily formulated for high throughput screening of GPCR modulators either with serum or without serum.